Human TLR5 Reporter HEK293 Cells (NF-κB)

NF-κB–SEAP reporter HEK293 cells expressing human TLR5

HEK-Blue™-hTLR5 cells were obtained by co-transfection of the human TLR5 gene and an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene into HEK293 cells.

The SEAP gene is placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Stimulation with a TLR5 ligand activates NF-κB and AP-1 which induce the production of SEAP.

Levels of SEAP can be easily determined with HEK-Blue™ Detection, a cell culture medium that allows for real-time detection of SEAP.

The parental cell line of HEK-Blue™ hTLR5 cells is HEK-Blue™ Null1 cells.

Figures

HEK-Blue™ hTLR5 cells were stimulated with various TLR and NOD agonists: Pam3CSK4 (100 ng/ml), Poly(I:C) (50 ng/ml), LPS-EB ultrapure (100 ng/ml), recFLA-ST (10 ng/ml), CL264 (1 μg/ml), CL097 (3 μg/ml), ssRNA40/LyoVec™ (5 μg/ml), ODN 2006 (10 μg/ml), C12-iE-DAP (100 ng/ml), L18-MDP (100 ng/ml), and TNF-α (100 ng/ml). After 18h incubation (24h incubation for CL264, C12-iE-DAP and L18-MDP ligands), NF-κB-induced SEAP activity was assessed using QUANTI-Blue™ by reading the OD at 655 nm.

HEK-Blue™ hTLR5 and HEK-Blue™ Null1 (control) cells were incubated in HEK-Blue™ Detection medium and stimulated with 100 ng/ml FLA-BS, 10 ng/ml FLA-ST Ultrapure, or 10 ng/ml recFLA-ST. After 24h incubation, the levels of NF-κB-induced SEAP were determined by reading the OD at 655 nm.

HEK-Blue™ hTLR5 cells were stimulated with increasing concentrations of TLR5 agonists. After 24h incubation, NF-κB-induced SEAP activity was assessed using HEK-Blue™ Detection by reading the OD at 655 nm. The response ratio was calculated by dividing the OD at 655 nm for the treated cells by the OD at 655 nm for the untreated cells.

Ligand	Origin	EC50	Response Ratio
FLA-BS	B.subtilis	15 +/- 5 ng/ml	29
FLA-ST	S.typhimurium	6 +/- 3 ng/ml	23

HEK-Blue™ TLR5 cells were pre-incubated for 1h with increasing concentrations of anti-hTLR5 IgA2, then stimulated with 5 ng/ml of FLA-ST Ultrapure for 18h. NF-kB-induced SEAP activity was assessed using QUANTI-Blue™ by reading the OD at 655 nm.

Specifications

Antibiotic resistance: blasticidin, Zeocin®.

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™.

Guaranteed mycoplasma-free.

This product is covered by a Limited Use License (See Terms and Conditions).

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Zeocin™ (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml)
• 1 pouch of HEK-Blue™ Detection (cell culture medium for real-time detection of SEAP)

Shipped on dry ice (Europe, USA, Canada, and some areas in Asia)

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